Genome-wide analysis of Claviceps paspali: insights into the secretome of the main species causing ergot disease in Paspalum spp.

BACKGROUND: The phytopatogen Claviceps paspali is the causal agent of Ergot disease in Paspalum spp., which includes highly productive forage grasses such as P. dilatatum. This disease impacts dairy and beef production by affecting seed quality and producing mycotoxins that can affect performance in feeding animals. The molecular basis of pathogenicity of C. paspali remains unknown, which makes it more difficult to find solutions for this problem. Secreted proteins are related to fungi virulence and can manipulate plant immunity acting on different subcellular localizations. Therefore, identifying and characterizing secreted proteins in phytopathogenic fungi will provide a better understanding of how they overcome host defense and cause disease. The aim of this work is to analyze the whole genome sequences of three C. paspali isolates to obtain a comparative genome characterization based on possible secreted proteins and pathogenicity factors present in their genome. In planta RNA-seq analysis at an early stage of the interaction of C. paspali with P. dilatatum stigmas was also conducted in order to determine possible secreted proteins expressed in the infection process. RESULTS: C. paspali isolates had compact genomes and secretome which accounted for 4.6-4.9% of the predicted proteomes. More than 50% of the predicted secretome had no homology to known proteins. RNA-Seq revealed that three protein-coding genes predicted as secreted have mayor expression changes during 1 dpi vs 4 dpi. Also, three of the first 10 highly expressed genes in both time points were predicted as effector-like. CAZyme-like proteins were found in the predicted secretome and the most abundant family could be associated to pectine degradation. Based on this, pectine could be a main component affected by the cell wall degrading enzymes of C. paspali. CONCLUSIONS: Based on predictions from DNA sequence and RNA-seq, unique probable secreted proteins and probable pathogenicity factors were identified in C. paspali isolates. This information opens new avenues in the study of the biology of this fungus and how it modulates the interaction with its host. Knowledge of the diversity of the secretome and putative pathogenicity genes should facilitate future research in disease management of Claviceps spp.

New Draft Genome Sequence of the Ergot Disease Fungus Claviceps paspali.

Here, we report a new draft genome sequence of an isolate of the ascomycete Claviceps paspali that is responsible for ergot disease in grasses of the Paspalum genus. This new draft genome sequence will provide useful data for evaluating intraspecies and interspecies genome variation in C. paspali and other Claviceps genus members.

Diversity of Claviceps paspali reveals unknown lineages and unique alkaloid genotypes.

Claviceps species affecting Paspalum spp. are a serious problem, as they infect forage grasses such as Paspalum dilatatum and P. plicatulum, producing the ergot disease. The ascomycete C. paspali is known to be the pathogen responsible for this disease in both grasses. This fungus produces alkaloids, including ergot alkaloids and indole-diterpenes, that have potent neurotropic activities in mammals. A total of 32 isolates from Uruguay were obtained from infected P. dilatatum and P. plicatulum. Isolates were phylogenetically identified using partial sequences of the genes coding for the second largest subunit of RNA polymerase subunit II (RPB2), translation elongation factor 1-α (TEF1), ß-tubulin (TUB2), and the nuc rDNA 28S subunit (28S). Isolates were also genotyped by randomly amplified polymorphic DNA (RAPD) and presence of genes within the ergot alkaloid (EAS) and indole-diterpene (IDT) biosynthetic gene clusters. This study represents the first genetic characterization of several isolates of C. paspali. The results from this study provide insight into the genetic and genotypic diversity of Claviceps paspali present in P. dilatatum and suggest that isolates from P. plicatulum could be considered an ecological subspecies or specialized variant of C. paspali. Some of these isolates show hypothetical alkaloid genotypes never reported before.

Current situation of pests targeted by Bt crops in Latin America.

Transgenic crops producing Bacillus thuringiensis- (Bt) insecticidal proteins (Bt crops) have provided useful pest management tools to growers for the past 20 years. Planting Bt crops has reduced the use of synthetic insecticides on cotton, maize and soybean fields in 11 countries throughout Latin America. One of the threats that could jeopardize the sustainability of Bt crops is the development of resistance by targeted pests. Governments of many countries require vigilance in measuring changes in Bt-susceptibility in order to proactively implement corrective measures before Bt-resistance is widespread, thus prolonging the usefulness of Bt crops. A pragmatic approach to obtain information on the effectiveness of Bt-crops is directly asking growers, crop consultants and academics about Bt-resistance problems in agricultural fields, first-hand information that not necessarily relies on susceptibility screens performed in laboratories. This type of information is presented in this report. Problematic pests of cotton and soybeans in five Latin American countries currently are effectively controlled by Bt crops. Growers that plant conventional (non-Bt) cotton or soybeans have to spray synthetic insecticides against multiple pests that otherwise are controlled by these Bt crops. A similar situation has been observed in six Latin American countries where Bt maize is planted. No synthetic insecticide applications are used to control corn pests because they are controlled by Bt maize, with the exception of Spodoptera frugiperda. While this insect in some countries is still effectively controlled by Bt maize, in others resistance has evolved and necessitates supplemental insecticide applications and/or the use of Bt maize cultivars that express multiple Bt proteins. Partial control of S. frugiperda in certain countries is due to its natural tolerance to the Bt bacterium. Of the 31 pests targeted and controlled by Bt crops in Latin America, only S. frugiperda has shown tolerance to certain Bt proteins in growers' fields, the most reliable indication of the status of Bt-susceptibility in most of the American continent.

Effect of pasture vs. concentrate feeding with or without antioxidants on carcass characteristics, fatty acid composition, and quality of Uruguayan beef.

Thirty Hereford steers were finished either on pasture (n=10) or concentrate (n=20) to determine dietary and antioxidant treatment effects on carcass characteristics, fatty acid composition, and quality of Uruguayan beef. Half of the steers finished on concentrate were supplemented with 1000 I.U. vitamin E head(-1) day(-1) for 100 days. Postmortem vitamin C was added to ground beef (0.05% v/w) displayed for 8 days at 2 °C. Carcasses from steers finished on concentrate had greater (P<0.05) carcass weight, conformation, degree of finishing, fat depth, and ribeye area than pasture finished animals. Carcasses from pasture-fed steers showed darker (P<0.05) longissimus color and yellower (P<0.05) fat at 24 h postmortem than concentrate-fed. Initial longissimus Warner-Bratzler shear force (WBSF) values were similar (P>0.05) between pasture- and concentrate-fed animals. However, beef from pasture-fed cattle had lower (P<0.05) WBSF values at 7 and 14 days postmortem. Longissimus α-tocopherol concentrations were greater (P<0.01) for pasture- and concentrate-fed animals that were supplemented with vitamin E compared to concentrate-fed. Steaks from pasture-fed and vitamin E supplemented cattle had similar (P>0.05) TBARS values, which were lower (P<0.05) than steaks from concentrate-fed steers during 21 days of display. Ground beef from vitamin E supplemented steers had the lowest TBARS values; whereas samples from pasture-fed animals had the lowest lipid stability with higher TBARS levels than other treatments. Vitamin C addition to ground beef did not (P>0.05) reduce lipid oxidation. Vitamin E supplementation of concentrate-fed cattle had no effect (P>0.05) on color stability of ground beef or steaks. The a(∗) (redness) and b(∗) (yellowness) values were higher (P<0.05) when vitamin C was added to ground beef. Longissimus fatty acid content of concentrate-fed animals was twofold greater (P<0.01) than pasture-fed. The percentages of C14:0, C16:0, and C18:1 fatty acids were higher (P<0.01) in the intramuscular fat of concentrate-fed steers, whereas pasture-fed cattle showed greater (P<0.01) proportions of C18:0, C18:2, C18:3, C20:4, C20:5, and C22:5. Total conjugated linoleic acid (CLA) and CLA isomer c9t11 were higher (P<0.01) for pasture- than concentrate-fed cattle. Vitamin E supplementation of concentrate-fed steers increased lipid stability of ground beef and steaks, but was unable to improve color stability; whereas vitamin C addition to ground beef increased color stability without altering lipid oxidation. Finishing cattle on pasture enhanced the unsaturated fatty acid profile of intramuscular fat in beef including CLA and omega-3 fatty acids.

Phytotoxic protein PcF, purification, characterization, and cDNA sequencing of a novel hydroxyproline-containing factor secreted by the strawberry pathogen Phytophthora cactorum.

A novel protein factor, named PcF, has been isolated from the culture filtrate of Phytophthora cactorum strain P381 using a highly sensitive leaf necrosis bioassay with tomato seedlings. Isolated PcF protein alone induced leaf necrosis on its host strawberry plant. The primary structure and cDNA sequence of this novel phytotoxic protein was determined, and BLAST searches of Swiss-Prot, EMBL, and GenBank(TM)/EBI data banks showed that PcF shared no significant homology with other known sequences. The 52-residue PcF protein, which contains a 4-hydroxyproline residue along with three S-S bridges, exhibits a high content of acidic sidechains, accounting for its isoelectric point of 4.4. The molecular mass of isolated PcF is 5,622 +/- 0.5 Da as determined by mass spectrometry and matches that calculated from the deduced amino acid sequence with cDNA sequencing. The cDNA sequence indicates that PcF is first produced as a larger precursor, comprising an additional N-terminal, 21-residue secretory signal peptide. Maturation of this protein involves the hydroxylation of proline 49, a feature that is unique among other known secreted fungal phytopathogenic proteins.